Immunohistochemistry

Forty-eight hours after the final injection (either LPS or saline), rats were anesthetized for tissue collection. This time point was also utilized for the acutely treated (i.e., single injection) rats, which were included to examine the interaction of food restriction on central and peripheral inflammation. Half of the rats were transcardially perfused with 200 ml each of ice-cold 1× phosphate-buffered saline (PBS; pH 7.4) and 4% paraformaldehyde (PFA; pH 7.4). Blood was collected with EDTA-treated syringes via the right ventricle prior to perfusion and transferred into plasma tubes (BD, Franklin Lakes, NJ, USA). Plasma tubes were inverted 6–8 times, placed on ice for approximately 30 min, and centrifuged at 1,600 g for 15 min at 4°C. Plasma was stored at −80°C until cytokine analysis. The brains were collected, stored in 4% PFA overnight, and then transferred to 30% sucrose. The brains were frozen in OCT compound and stored at −80°C until slicing as 40 μm-thick coronal sections on a cryostat (Microm, Waltham, MA, USA). These sections were stored at −20°C in cryoprotectant solution (15% glucose, 30% ethylene glycol, 0.04% sodium azide, 0.05 M PBS) until later processing using fluorescent immunohistochemistry (IHC) to evaluate the impact of age and peripheral inflammation on astrocytic and microglial activation.

To evaluate the presence of neuroinflammation after peripheral LPS treatment, prefrontal coronal sections were tagged for glial fibrillary acidic protein (GFAP) or ionized calcium binding adaptor molecule (Iba-1) costained with CD68 as astrocyte and microglial markers, respectively. Free-floating sections were rinsed of cryoprotectant solution, incubated in antigen unmasking solution (Vector, Burlingame, CA, USA) at 95°C for 7 min, and rinsed 3 × 5 min in 1× PBS. Sections were blocked in 10% donkey serum, 0.3% Triton, and PBS for 2 h and incubated overnight in primary antibody solution, including 1% bovine serum albumin (BSA), 0.3% triton, and PBS (chicken anti-GFAP: 1:500, ABCam, Cambridge, MA, USA; goat anti-Iba-1: 1:500, ABCam; mouse anti-rat CD68: 1:200, BioRad, Hercules, CA, USA). The sections were rinsed and incubated for 2 h in secondary antibody solution of 1% BSA in PBS (Alexa Fluor 488 donkey anti-chicken, 1:500, Jackson, Bar Harbor, ME, USA; Alexa Fluor 488 donkey anti-goat, 1:500, Life Technologies, Waltham, MA, USA; Alexa Fluor 594 donkey anti-mouse, 1:800, Life Technologies, Waltham, MA, USA). After a final rinse, slices were mounted onto gelatin-coated slides and coverslipped with mounting medium containing DAPI (Vector).

Three representative sections of the medial PFC, specifically the prelimbic cortex (A/P range: +3.7 to +2.0), were processed for each rat (N = 4/group for behaviorally performing rats; N = 3–6/group for acutely treated rats) for each fluorescent stain. Images were captured at 200× magnification on a Leica DM2500 microscope (Wetzlar, Germany), equipped with a Retiga 4000R camera (QImaging, Surrey, BC, Canada) with QCapture Pro7 software (QImaging, Surrey, BC, Canada). Utilizing NIH ImageJ, the size and number of GFAP+ (astrocytes) and Iba-1+ cells (microglia) were measured. Briefly, the image was converted from pixels to micrometer (2.28 pixels/μm for 200× magnification), enhanced to reduce background and improve visualization of the positively-stained cells, and analyzed for count and size. Colocalization of Iba-1 and CD68, which represented [(Iba-1+/CD68+)/Iba-1+ only]*100, was assessed using Adobe Photoshop (San Jose, CA, USA) for 100 Iba-1-positive cells in the prelimbic cortex of each rat.